The impact of chronic hepatitis C infection on cholesterol metabolism in PBMCs is associated with microRNA-146a expression.

Chronic hepatitis C (CHC) infection is known to induce important changes in host cholesterol metabolism. MicroRNAs (miRNAs) regulate the expression of many genes and, in consequence, control various processes, including human metabolism and response to viral infection. Recently, the alteration of the immune-associated miR-146a, which is abundantly present in peripheral blood mononuclear cells (PBMCs), was found in some viral infections. The study aimed to analyse the influence of hepatitis C virus (HCV) infection on miR-146a expression in PBMCs in vivo and in vitro, as well as to assess the possible impact of miR-146a alteration on the intracellular cholesterol level in PBMCs. Blood samples collected from 42 healthy donors and 72 CHC patients were the source of materials. HCV RNA, intracellular cholesterol level and miR-146a expression were determined in PBMCs, as well as HCV genotype and interferon (IFN)α concentration in sera. The influence of miR-146a inhibition on cholesterol expression in PBMCs was analysed in vitro after transient cell transfections with mirVana™ anti-miR-146a Inhibitor. Our data demonstrated an alteration of miR-146a and intracellular cholesterol expression in PBMCs and of IFNα concentration in sera of genotype 1, HCV-infected patients compared to the healthy donors. Also, in cultured PBMCs, miR-146a expression and intracellular cholesterol level were significantly decreased in CHC patients compared to the healthy donors. In vitro blockage of miR-146a expression in PBMCs of CHC patients greatly impaired intracellular cholesterol expression. In these conditions, miR-146a expression was positively correlated with the intracellular cholesterol level. These results suggest that genotype 1 HCV infection may alter miR-146a expression in PBMCs and, consequently, contribute to the observed dysregulation of cholesterol synthesis.

The altered expression of α1 and ß3 subunits of the gamma-aminobutyric acid A receptor is related to the hepatitis C virus infection.

The modulation of the gamma-aminobutyric acid type A (GABA A) receptors activity was observed in several chronic hepatitis failures, including hepatitis C. The expression of GABA A receptor subunits α1 and ß3 was detected in peripheral blood mononuclear cells (PBMCs) originated from healthy donors. The aim of the study was to evaluate if GABA A α1 and ß3 expression can also be observed in PBMCs from chronic hepatitis C (CHC) patients and to evaluate a possible association between their expression and the course of hepatitis C virus (HCV) infection. GABA A α1- and ß3-specific mRNAs presence and a protein expression in PBMCs from healthy donors and CHC patients were screened by reverse transcription polymerase chain reaction (RT-PCR) and Western blot, respectively. In patients, HCV RNA was determined in sera and PBMCs. It was shown that GABA A α1 and ß3 expression was significantly different in PBMCs from CHC patients and healthy donors. In comparison to healthy donors, CHC patients were found to present an increase in the expression of GABA A α1 subunit and a decrease in the expression of ß3 subunit in their PBMCs. The modulation of α1 and ß3 GABA A receptors subunits expression in PBMCs may be associated with ongoing or past HCV infection.

Expression of microRNA-155 precursor in peripheral blood mononuclear cells from Hepatitis C patients after antiviral treatment.

Chronic hepatitis caused by Hepatitis C virus (HCV) is the main source of liver cirrhosis, hepatocellular carcinoma, and extra-hepatic diseases. After treatment-induced resolution of hepatitis C, the persistence of HCV RNA in serum and peripheral blood mononuclear cells (PBMCs) is often observed. An expression of the precursor of microRNA-155 (miR-155) called BIC can be the factor responsible for a course of HCV infection. Therefore, we assessed the relationship between BIC expression and HCV RNA status in sera and PBMCs samples of 64 hepatitis C patients treated with interferon alpha(IFN-alpha)+ribavirin. High expression of BIC in PBMCs was determined in 100% of patients that harbored HCV RNA in serum and PBMCs. Further, we found that 83% of PBMCs samples were BIC-positive in a group of patients that eliminated HCV RNA only from serum. The lowest expression of BIC was found in patients that eliminated HCV RNA from both serum and PBMCs.

Effect of interferon alfa and ribavirin treatment on hepatitis C virus RNA in serum and peripheral blood mononuclear cells in children with chronic hepatitis C.

UNLABELLED: The correlations between the severity of hepatic lesions, age, gender, HBV co-infection and negativisation of HCV-RNA from serum and peripheral blood mononuclear cells (PBMC) after treatment of chronic hepatitis C (CHC) were analysed. 41 children (11 F/ 30 M), aged 5-16 years (mean 10 +/- 2.8), were treated with IFN-alpha and ribavirin for 12 months. Sustained negativisation of HCV-RNA from serum was achieved in 25 patients (61%), in 3 (7%) it reappeared after treatment, and in 13 (32%) it was ineffective. Clearance of HCV did not correlate with age (p = 0.65), sex (p = 0.13), past HBV infection (n = 22 anti-HBc +) (p = 0.24), maximum pre-treatment ALT activity (p = 0.06), grade of inflammation (p = 0.33) or stage of fibrosis (p = 0.9) in liver biopsy. It was achieved in 6/16 children previously resistant to IFN-a monotherapy and in 19/25 naive (p = 0.017). HCV-RNA was detected in PBMC in 9/24 (37%) seronegative children and in 1/21 (5%) in comparative group of seronegative adults; p = 0.004. Persistence of HCV-RNA in PBMC after combined treatment occurred in 5/10 (50%) patients resistant to previous IFN-alpha monotherapy, 6/35 (20%) of them cleared HCV from PBMC (p = 0.04). CONCLUSIONS: Age and gender, infection route, history of HBV infection or severity of histopathologic liver lesions had no influence on the efficacy of treatment with IFN-alpha and ribavirin. Clearance of HCV from serum and from PBMC occurs less frequently in patients previously resistant to IFN-alpha. Children with CHC require longitudinal observation after successful antiviral treatment as in 37% of those considered to be free from the virus by ordinary measures, HCV-RNA was found in PBMC.

The effect of interleukin-6 on hepatitis B virus replication in peripheral blood mononuclear cells in vitro.

Although the major target organ for hepatitis B virus (HBV) is the liver, the possibility of infection of peripheral blood mononuclear cells (PBMCs) with HBV has also been reported. This study was performed to analyze the course of HBV infection of PBMCs and to investigate the influence of interleukin-6 (IL-6) on the efficiency of infection of PBMCs with HBV in vitro. PBMCs isolated from a healthy donor were infected by exposing to a HBsAg-, HBeAg-positive serum in the presence or absence of exogenous IL-6. The efficiency of infection was estimated by HBV DNA determination in the cells and medium in the course of infection. The results of this study show that the presence of IL-6 during the PBMCs infection with HBV increased the efficiency of this infection.

Nonenveloped nucleocapsids of hepatitis C virus in the serum of infected patients.

One of the characteristics of hepatitis C virus (HCV) is the high incidence of persistent infection. HCV core protein, in addition to forming the viral nucleocapsid, has multiple regulatory functions in host-cell transcription, apoptosis, cell transformation, and lipid metabolism and may play a role in suppressing host immune response. This protein is thought to be present in the bloodstream of the infected host as the nucleocapsid of infectious, enveloped virions. This study provides evidence that viral particles with the physicochemical, morphological, and antigenic properties of nonenveloped HCV nucleocapsids are present in the plasma of HCV-infected individuals. These particles have a buoyant density of 1.32 to 1.34 g/ml in CsCl, are heterogeneous in size (with predominance of particles 38 to 43 or 54 to 62 nm in diameter on electron microscopy), and express on their surface epitopes located in amino acids 24 to 68 of the core protein. Similar nucleocapsid-like particles are also produced in insect cells infected with recombinant baculovirus bearing cDNA for structural HCV proteins. HCV core particles isolated from plasma were used to generate anti-core monoclonal antibodies (MAbs). These MAbs stained HCV core in the cytoplasm of hepatocytes from experimentally infected chimpanzees in the acute phase of the infection. These chimpanzees had concomitantly HCV core antigen in serum. These findings suggest that overproduction of nonenveloped nucleocapsids and their release into the bloodstream are properties of HCV morphogenesis. The presence of circulating cores in serum and accumulation of the core protein in liver cells during the early phase of infection may contribute to the persistence of HCV and its many immunopathological effects in the infected host.

[Evaluation of persistence of IFN-alpha treatment response in chronic hepatitis C patients according with HCV-RNA presence in PBMC]. / Ocena trwalosci odpowiedzi na leczenie IFN-alpha chorych na przewlekle zapalenie watroby typu C na podstawie obecnosci HCV-RNA w komórkach jednojadrowych krwi obwodowej.

BACKGROUND: The current criterion of cure of chronic hepatitis C (CHC) is the absence of HCV-RNA in hepatocytes and peripheral blood mononuclear cells (PBMC), as these cells are considered for the place of protracted viral replication. PURPOSE: Evaluation of HCV-RNA presence in serum, freshly isolated PBMC and cultured PBMC, taken from patients responding to interferon alfa2b therapy, after 42 months of follow-up. Fifty four adults with CHC was treated with interferon alfa-2b (IFN-alpha 2b). Evaluation of HCV-RNA presence in serum were performed using RT-PCR method, before the therapy, and after 6 and 18 months of follow-up. All patients received interferon alpha-2b for 6 months. After 18 months of follow-up, results of treatment (ALT activity and presence of HCV-RNA in serum) were retrospectively analyzed. In 10 of 54 patients (18.5%) normalization of ALT activity and negativization of HCV-RNA in serum were observed. After 42 months of follow-up, in these 10 patients, the presence of HCV-RNA in serum, in freshly isolated PBMC and cultured PBMC, were evaluated. RESULTS: After 42 months of follow-up, HCV-RNA was not found in serum of any patient. In 3 of 10 patients HCV-RNA was present in freshly isolated PBMC. After 3-day-culture of PBMC, HCV-RNA was found in another patient. After 6 days of culture, results of RT-PCR test were negative in all cases. CONCLUSIONS: Absence of HCV-RNA in serum as well as in freshly isolated PBMC after 42 months of follow-up after the treatment of CHC, is not synonymous with eradication of virus. Three-day-culture of PBMC may be a valuable method, allowing for detection of subtreshold amounts of HCV. Longer, 6-day-culture seems to be invaluable diagnostically.

The newly synthetized pyridobenzoxazocynone inhibits HIV-1 reverse transcriptase activity.

We synthetized pyridobenzoxazocynone that differs in the enlarged eight-membered heterocyclic system from the basic structure of pyridobenzoxazepinones a known class of non-nucleoside HIV-1 reverse transcriptase inhibitors. Pyridobenzoxazocynone hydrochloride was found to inhibit HIV-1 reverse transcriptase activity. At concentration 0.35 microM the enzyme activity decreased by 64 +/- 14%. Higher concentrations of pyridobenzoxazocynone hydrochloride completely abolished the enzyme activity expressed as radioactivity of acid insoluble products. These results suggest that pyridobenzoxazocynones may represent a new class of HIV-1 reverse transcriptase inhibitors.

Oxidative burst response of neutrophils primed with PreS1 antigen of hepatitis B virus in patients with chronic hepatitis B and convalescents.

The pathogenesis of liver damage in the course of hepatitis B virus (HBV) infection depends on the host's specific and non-specific immune response to various viral antigens. The role of polymorphonuclear neutrophils (PMN) in the natural immune reaction and during secondary microbial infections is well documented. Increased free radical production is associated with many pathological conditions such as shock, ischaemia or chronic inflammatory diseases. We studied the oxidative metabolism of neutrophils in patients with chronic HBV and after recovery (convalescents). The effect of the PreS1 fragment of HBV antigen on some neutrophil functions in vitro was also examined. There were significant differences in the values of spontaneous and stimulated oxidative burst of neutrophils, measured using luminol-chemiluminescence, in patients with HBV when compared with the convalescents. PreS1 antigen did not by itself induce the respiratory burst in human neutrophils but it potentiated their response to a second stimulus. Hence we observed a priming of neutrophils, for an enhanced respiratory burst, by PreS1 antigen.

Effect of pre-S1 antigen on human lymphocyte proliferative responses.

The peripheral blood mononuclear cells (PBMCs) from patients acutely infected with HBV and recovered completely (n = 20), patients with chronic hepatitis B infection (CHB)- (n = 10) and HBsAg-positive carriers (n = 9) and healthy individuals (n = 8) were studied for their in vitro proliferative response to a synthetic pre-S1(20-49)x4 antigen. PBMCs from convalescents showed significant proliferative response in the presence of synthetic pre-S1 antigen. PBMCs from CHB- and HBsAg-positive exhibited reduced proliferative response not only to Pre-S1 antigen but also to nonspecific mitogens. This study suggests that the immune recognition of pre-S1 antigen and response of PBMCs to the pre-S1 antigen may be an important part of the normal human response to HBV infection. Failure to clear the HBV infection with development of the chronic carrier state may be caused by the lack of an efficient pre-S1 antigen-specific response.

Expression and characterization of the multiplied, recombinant preS1 antigen of hepatitis B virus.

The amino acid sequence encoded by the preS1 region of hepatitis B virus genome is expressed on the surface of virions and subviral particles. The preS1 region is involved in the recognition of specific receptors responsible for the attachment of HBV to the host cell. The cell receptor binding site was assigned to the preS1 (20-47 aa) fragment. In order to obtain a large quantity of preS1 binding domains of HBV the expression vector pWX4 was constructed. It contains four tandemly joined DNA sequences, each coding for preS1 (20-49 aa), fused with the 3' end of a DNA fragment coding for 450 aa of beta-galactosidase. E. coli cells transformed with this vector produce fusion protein beta-gal-preSlx4 in the form of inclusion bodies. Owing to the specific trypsin digestion, the preSlx4 domain was cleaved from the fusion protein. The resulting product, a 16 kDa protein, was isolated and purified by anion exchange chromatography. The presence of four Asp-Pro bonds in this sequence and the primary structure of the first 28 N-terminal amino acids were determined. Following the confirmation of the antigenic properties, the recombinant preS1 protein was used for detection of the anti-preS1 response in sera from HBV infected patients.

Effect of Pre-S1 protein on the long-term cultures of human lymphocytes after hepatitis B immunization.

Long-term cultured T-cells, reactive to Pre-S1 protein, were developed from peripheral blood mononuclear cells (PBMC) of individuals after recovery from hepatitis B infection and of vaccine recipients by in vitro Pre-S1 protein stimulation in the presence of IL-2. The proliferative responses to Pre-S1 protein and functional activities of cultured T-cells were characterized.

Expression of multivalent pre-S1 antigen of hepatitis B virus in Escherichia coli (synthetic oligodeoxynucleotides, surface antigens, recombinant DNA, fusion protein, beta-galactosidase).

The nucleotide sequence encoding 30 amino acids (aa) of the pre-S1 envelope region of the human hepatitis B virus has been constructed from twenty chemically synthesized oligodeoxynucleotides by simultaneous ligation. The DNA fragment containing four repeated sequences encoding the pre-S1 region (aa 20-49) has been inserted into the lacZ gene of the plasmid pWR450.1, yielding the recombinant pWX4 plasmid. The Escherichia coli DH5 strain transformed with pWX4 produces a beta-galactosidase-[-pre-S1(20-49) x 4] fusion protein. The hybrid protein containing 127 aa of repeated pre-S1 region has been isolated from Escherichia coli as inclusion bodies and purified by anion exchange chromatography. The antigenic properties of this fusion protein were confirmed by immunoblotting with pre-S1-specific monoclonal antibodies.